The goal of our studies is to understand the biochemical pathways and to identify key proteins (enzymes) that are involved in the activation and the effector phase of such important immune responses, like T-cell- mediated cytotoxicity and lymphokine secretion. It is expected,that such knowledge will enable us to selectively manipulate different functional responses of T-cells with specifically designed immunomodulators. We attempt to reach this goal by taking advantage of the (A) extensive knowledge of the structure of the well known cAMP-Dependent Protein Kinases (PKA) and by (B) developing new approaches for the isolation of novel, functionally important protein kinases. A) In experiments designed to directly evaluate the role of the PKA's catalytic (C alpha) subunit in Cytotoxic T-Lymphocytes (CTL) effector functions, cells were pretreated with antisense oligomers complementary to mRNA for the C alpha subunit of PKA. In another approach the role of PKA in interleukin-2 (IL-2) production was studied by transfection of T-cells with C alpha-inhibiting regulatory subunit (RIalpha) of PKA with dominant negative mutation and by using antisense C alpha mRNA oligodeoxynucleotides. The obtained results suggest,that the free C alpha subunit of PKA down- regulates TCR-triggered, protein-synthesis independent responses like cytotoxicity and exocytosis, but is required for the protein synthesis- dependent processes like interferon or IL-2 secretion by T-lymphocytes. Thus, these results point to PKA as an attractive target for the immunomodulation. B) Identification of Ectoprotein Kinases in Lymphocytes. While pursuing our "extracellular ATP" hypothesis of the cell-mediated cytotoxicity, we demonstrated the presence and characterized highly active and extracellularly located protein kinases (ectokinases). Their isolation and cloning promises to provide a very novel set of potentially functionally important proteins.